U.S. patent application number 11/547405 was filed with the patent office on 2008-05-08 for microchip and method for detecting molecules and molecular interactions.
This patent application is currently assigned to Nanyang Technological University. Invention is credited to Changming Li.
Application Number | 20080108095 11/547405 |
Document ID | / |
Family ID | 39398923 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080108095 |
Kind Code |
A1 |
Li; Changming |
May 8, 2008 |
Microchip and Method for Detecting Molecules and Molecular
Interactions
Abstract
A microchip with flow-through inlet (104) and outlet (106)
channels and test channels (108). The test channels (108) are in
fluid communication with the inlet (104) and outlet (106) channels,
through inlets (114) and outlets (116) respectively. Each test
channel (108) has one test site therein for detection of specific
molecules or molecular interactions. The inlet (114) in a test
channel (108) is elevated from the outlet (116) of the test channel
(108) and the outlet (116) is elevated from a fluid level (124) in
the outlet channel (106). Back diffusion from outlet channel (106)
to the test channels (108) and from the test channels (108) to the
inlet channel (104) can thus be inhibited to reduce or eliminate
cross-interference between different test sites. The microchip can
be useful as a flow-through high density enzyme immunoassay array
device with high-throughput.
Inventors: |
Li; Changming; (Singapore,
SG) |
Correspondence
Address: |
DINSMORE & SHOHL LLP
ONE DAYTON CENTRE, ONE SOUTH MAIN STREET, SUITE 1300
DAYTON
OH
45402-2023
US
|
Assignee: |
Nanyang Technological
University
Singapore
SG
|
Family ID: |
39398923 |
Appl. No.: |
11/547405 |
Filed: |
April 1, 2005 |
PCT Filed: |
April 1, 2005 |
PCT NO: |
PCT/SG05/00112 |
371 Date: |
October 2, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60558116 |
Apr 1, 2004 |
|
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60558118 |
Apr 1, 2004 |
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Current U.S.
Class: |
435/7.93 ;
422/68.1; 435/7.92; 73/61.41 |
Current CPC
Class: |
B01L 2400/0457 20130101;
B01L 2300/0874 20130101; B01L 2300/0867 20130101; G01N 33/5438
20130101; B01L 2300/0864 20130101; B01L 2400/0487 20130101; B01L
2200/141 20130101; G01N 33/54366 20130101; B01L 2300/0816 20130101;
B01L 2300/0636 20130101; B01L 3/502746 20130101; B01L 2200/027
20130101; B01L 2300/0645 20130101; B01L 2300/0851 20130101 |
Class at
Publication: |
435/7.93 ;
422/68.1; 435/7.92; 73/61.41 |
International
Class: |
C12Q 1/00 20060101
C12Q001/00; B01J 19/00 20060101 B01J019/00; G01N 33/00 20060101
G01N033/00 |
Claims
1. A microchip comprising: a substrate; an inlet channel in said
substrate; an outlet channel in said substrate for guiding fluid
below a fluid level; a plurality of test channels in said substrate
for guiding fluid flow from said inlet channel to said outlet
channel, each one of said test channels having an inlet for fluid
communication with said inlet channel and an outlet for fluid
communication with said outlet channel, said inlet elevated from
said outlet for inhibiting back flow through said inlet, said
outlet elevated from said fluid level for inhibiting back flow
through said outlet; and one test site in each one of said test
channels, for detection of at least one of a specific molecule and
a molecular interaction at said test site.
2. The microchip of claim 1, wherein each one of said channels has
a substantially flat bottom, said bottom of said inlet channel
elevated from bottoms of said test channels, said bottoms of said
test channels elevated from said bottom of said outlet channel.
3. The microchip of claim 1, wherein at least one of said test
channels comprises a well for holding fluid therein.
4. The microchip of any one of claim 1, wherein said inlet and
outlet channels are adapted to allow a liquid to flow at a greater
rate in said outlet channel than in said inlet channel.
5. The microchip of any one of claim 1, wherein each one of said
test sites comprises a surface suitable for immobilizing specific
molecules thereon.
6. The microchip of any one claim 1, further comprising probe
molecules immobilized at each one of said test sites, said probe
molecules having specific affinity to selected target
molecules.
7. The microchip of claim 6, wherein different probe molecules are
immobilized at different ones of said test sites for detecting
different target molecules.
8. The microchip of any one of claim 1, further comprising a
plurality of electrodes, one of said electrodes at each one of said
test sites.
9. The microchip of claim 8, wherein at least one of said test
channels comprises a narrowed section proximate said test site in
said at least one test channel for guiding fluid towards said test
site.
10. The microchip of claim 8, wherein said plurality of electrodes
are first electrodes, said microchip further comprising a plurality
of second electrodes, one of said second electrodes in each one of
said test channels.
11. The microchip of claim 10, wherein said first electrodes are
interconnected and said second electrodes are interconnected, such
that each pair of said electrodes in each one of said test channels
is uniquely addressable.
12. The microchip of claim 10, further comprising probe molecules
immobilized proximate said first electrodes.
13. The microchip of any one of claim 1, wherein said test channels
extend substantially in parallel.
14. The microchip of any one of claim 1, further comprising a
dilution channel formed in said substrate, said dilution channel in
fluid communication with said inlet channel and having a first
inlet for reception of a first fluid and at least one second inlet
for reception of a second fluid to dilute said first fluid.
15. The microchip of claim 14, wherein said at least one second
inlet comprises a plurality of second inlets.
16. The microchip of any one of claim 1, wherein said substrate
comprises polydimethylsiloxane (PDMS).
17. The microchip of claim 16, wherein a PDMS surface in each one
of at least one of said plurality of test sites is coated with
aminopropyltriethoxysilane (APTES), thus forming a PDMS-APTES
surface.
18. The microchip of claim 17, wherein probe molecules are
covalently immobilized on said PDMS-APTES surface through
cross-linkers.
19. The microchip of claim 18, wherein said cross-linkers comprise
at least one of glutaraldehyde and
1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide.
20. The microchip of any one of claim 1, wherein at least one of
said channels is adapted for connection to at least one of a pump
and a valve for regulating fluid flow.
21. A method of detecting molecules or molecular interaction,
comprising: providing a microchip having a substrate, an inlet
channel in said substrate, an outlet channel in said substrate, a
plurality of test channels in said substrate each having an inlet
for fluid communication with said inlet channel and an outlet for
fluid communication with said outlet channel, said inlet elevated
from said outlet, and one test site in each one of said plurality
of test channels; introducing a fluid to said test channels through
said inlet channel; inhibiting back flow of said fluid through said
inlets and outlets by allowing overflow of said fluid from said
test channels to said outlet channel through said outlets and
maintaining the fluid level in said outlet channel below said
outlets, thus inhibiting diffusion of molecules in said fluid from
one of said test sites to another one of said test sites; and after
said fluid has been introduced to said test channels, detecting at
least one of a molecule and a molecular interaction at a test site
within said plurality of test sites.
22. The method of claim 21, wherein said detecting comprises
detecting a signal indicative of at least one of the presence of
said molecule and the occurrence of said molecular interaction,
said signal comprising at least one of optical, electrical,
magnetic, radiation, and electromagnetic signals.
23. The method of claim 21, wherein probe molecules are immobilized
at said test site for selectively capturing at least one of target
molecules and reporter molecules.
24. The method of any one of claim 21, wherein said at least one of
said molecule and molecular interaction is detected at said test
site by enzyme immunoassaying (EIA).
25. The method of claim 24, wherein said EIA comprises
enzyme-linked immunosorbent assaying (ELISA).
26. The method of claim 24, wherein said EIA comprises
electrochemical EIA (EIA-EC).
27. The method of any one of claim 26, wherein said EIA comprises
one of direct EIA, competitive EIA, competitive inhibition EIA, and
sandwich EIA:
28. The method of claim 27, wherein said detecting comprises
detecting an electrical signal and said microchip comprises a
plurality of electrodes, at least one of said electrodes at each
one of said test sites for detecting said electric signal.
29. The method of claim 28, wherein said electric signal comprises
at least one of a current, a voltage, and impedance.
30. The method of any one of claim 29, wherein said microchip
further comprises a dilution channel in fluid communication with
said inlet channel and having a first inlet and at least one second
inlet, said introducing a fluid comprising feeding said fluid into
said dilution channel through said first inlet and feeding a
dilution fluid into said dilution channel through said at least one
second inlet so that a diluted fluid is received in said inlet
channel and thus said test channels.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit and priority from U.S.
provisional patent application No. 60/558,116 filed Apr. 1, 2004
and U.S. provisional patent application No. 60/558,118 filed Apr.
1, 2004, the contents of each of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to detection of
molecules and molecular interactions, and more particularly to
microchips and methods for detecting molecules and their
interactions.
BACKGROUND
[0003] Detection of biomolecules or their interactions has
applications in many biological and biochemical industries. For
example, it has been widely used for medical diagnostic
applications.
[0004] Known detection devices of biomolecules and their
interactions include microchips or biochips with arrays of
addressable test sites thereon. Such devices are powerful
analytical tools because hundreds or thousands of unique test sites
can be analyzed simultaneously, with high throughput. In a typical
conventional microchip, target molecules can be captured and
immobilized at different spots on the microchip for detection.
Different test sites may be used to detect different target
molecules or the same molecules in different samples. Various
techniques may be used to detect the target molecules. For example,
the target molecules may be detected optically or electrically.
Further, biomolecules or biomolecular interactions may be detected
by the enzyme immunoassay (EIA) or (ed immunosorbent assay (ELISA)
techniques, with extremely high ind specificity.
[0005] However, conventional microchips suffer certain drawbacks.
For example, in known devices the test sites are often immersed in
liquid during detection and cross-interference between different
test sites can occur due to diffusion of the molecules to be
detected from one test site to another. Such cross-interference can
lead to false or inaccurate detection results. The molecules to be
detected can be either the target molecules or reaction products
which are to be detected to indicate the presence of the target
molecules.
[0006] A known approach to avoid cross-interference due to
diffusion is to increase the distance between different test sites,
to reduce the effect of diffusion. For example, Ying Ding et al.
disclosed a device based on the EIA electrochemical technique
(EIA-EC) in "Feasibility studies of simultaneous multianalyte
amperometric immunoassay based on spatial resolution", Journal of
Pharmaceutical and Biomedical Analysis, (1999), vol. 19, p. 153,
the contents of which are incorporated herein by reference. In this
detection device, each test site has a working electrode and the
distance between two adjacent electrodes is 2.5 mm. This spatial
separation is found to be effective for avoiding cross-interference
caused by diffusion when the detection measurement is completed
within a certain time period after introducing the enzyme
substrate. However, such an approach also has some drawbacks. One
problem is that only a small number of test sites can be formed on
a microchip device when the distance between adjacent electrodes is
so large.
[0007] An alternative known approach of optical ELISA or EIA is to
provide an array of isolated test wells in a chip to avoid
cross-interference caused by diffusion. However, these well-based
microchips have their own limitations. One problem is that
well-density is limited, due to either manufacturing or operation
requirements, so that it is difficult to form high-density test
sites. Another problem is that a well-based microchip does not
allow flow-through operation, thus making automated operation
difficult. For example, automated sampling, washing, or dilution on
such a chip may require expensive equipments such as robotic
devices.
[0008] Thus, there remains a need of improved devices and methods
for detection of molecules and molecular interactions.
SUMMARY OF THE INVENTION
[0009] In summary, a microchip with flow-through inlet and outlet
channels and test channels is disclosed. The test channels are in
fluid communication with the inlet and outlet channels, through
inlets and outlets respectively. Each test channel has one test
site therein for detection of specific molecules or molecular
interactions. The inlet in a test channel is elevated from the
outlet of the test channel and the outlet is elevated from a fluid
level in the outlet channel.
[0010] Advantageously, back flow or diffusion from the outlet
channel to the test channels and from the test channels to the
inlet channel can be inhibited to reduce or eliminate
cross-interference between different test sites.
[0011] Accordingly, an aspect of the present invention relates to a
microchip. The microchip comprises a substrate, an inlet channel in
the substrate, an outlet channel in the substrate for guiding fluid
below a fluid level, and a plurality of test channels in the
substrate for guiding fluid flow from the inlet channel to the
outlet channel. Each one of the test channels has an inlet for
fluid communication with the inlet channel and an outlet for fluid
communication with the outlet channel. The inlet is elevated from
the outlet for inhibiting back flow through the inlet. The outlet
is elevated from the fluid level for inhibiting back flow through
the outlet. One test site is in each one of the test channels for
detection of a specific molecule or molecular interaction at the
test site.
[0012] Another aspect of the present invention relates to a method
of detecting molecules or molecular interaction. In this method, a
microchip is provided. The microchip has a substrate, an inlet
channel in the substrate, an outlet channel in the substrate, a
plurality of test channels in the substrate each having an inlet
for fluid communication with the inlet channel and an outlet for
fluid communication with the outlet channel. The inlet is elevated
from the outlet. One test site is in each one of the plurality of
test channels. A fluid is introduced to the test channels through
the inlet channel. Back flow through the inlets and outlets is
inhibited by allowing overflow of the fluid from the test channels
to the outlet channel through the outlets and maintaining the fluid
level in the outlet channel below the outlets, thus inhibiting
diffusion of molecules in the fluid from one of the test sites to
another one of the test sites. After the fluid has been introduced
to the test channels, a molecule or molecular interaction is
detected at a test site within the plurality of test sites.
[0013] In optical detection, the probe molecules may be directly
immobilized on the bottom surface of the detection channel, i.e.
detection well; in electronic detection, two (working and counter
electrodes) or three electrodes (working, counter and reference
electrodes are fabricated in the detection channels, where in all
working and counter electrodes can be interconnected respectively
for addressable electronic detection.
[0014] Other aspects and features of the present invention will
become apparent to those of ordinary skill in the art upon review
of the following description of specific embodiments of the
invention in conjunction with the accompanying Figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In the figures, which illustrate exemplary embodiments of
the invention,
[0016] FIG. 1A is a plan view of a microchip, exemplary of an
embodiment of the present invention;
[0017] FIG. 1B is a cross-sectional view of the microchip of FIG.
1A taken along the line B-B;
[0018] FIG. 2A is a plan view of another microchip exemplary of a
further embodiment of the present invention;
[0019] FIG. 2B is a partial cross-sectional view of the microchip
of FIG. 2A taken along the line B-B;
[0020] FIG. 3A is a partial cross-sectional view of a variation of
the microchip of FIGS. 2A and 2B;
[0021] FIG. 3B is a partial plan view of the microchip of FIG.
3A;
[0022] FIG. 4 is a partial plan view of a variation of the
microchip of FIG. 3A;
[0023] FIGS. 5A and 5B are schematic diagrams illustrating
exemplary enzyme immunoassay techniques;
[0024] FIG. 6A is a partial image of a mold for forming a
microchip; and
[0025] FIG. 6B is a partial image of a microchip formed from the
mold of FIG. 6A.
DETAILED DESCRIPTION
[0026] FIGS. 1A and 1B illustrate a microchip or microfluidic
biochip 100, exemplary of an embodiment of the present
invention.
[0027] Biochip 100 includes a substrate 102. An inlet channel 104,
an outlet channel 106, and a plurality of test channels 108 are
formed in substrate 102. The test channels 108 may be substantially
parallel to each other. Inlet channel 104 is in fluid communication
with, and can receive fluid from, a dilution channel 110. Dilution
channel 110 has multiple inlets 112A to 112D (also collectively and
individually referred to as 112), each for introducing or feeding a
fluid into dilution channel 110. Each test channel 108 has an inlet
114 and an outlet 116. Each test channel 108 is in fluid
communication with inlet channel 104 through inlet 114 and with
outlet channel 106 through outlet 116. While four test channels 108
are depicted in FIG. 1A, the number of test channels 108 may vary
depending on the particular application and manufacturing
considerations. Each test channel 108 has one test site therein for
detection of a specific molecule or molecular interaction at the
test site, as will be further described below.
[0028] As depicted in FIG. 1B, a test channel 108 has a bottom 118
and an overflow wall 120 which forms the lower end of outlet 116.
However, as will become apparent, wall 120 is optional and can be
omitted. With wall 120, a test well is formed in each channel 108.
The well structure may be advantageous in some applications, as
will become clear below.
[0029] As can be better seen in FIG. 1B, outlet channel 106 has a
bottom 122 below outlet 116 for guiding fluid below a fluid level,
such as indicated by the dashed line 124. For each test channel
108, inlet 114 is elevated from outlet 116, which is in turn
elevated from the fluid level 124 in outlet channel 106. As can be
understood, inlet channel 104 can thus guide fluid at a fluid level
above the fluid level in each test channel 108. A test channel 108
can hold fluid at a fluid level up to outlet 116, as indicated by
the dashed line 126 in FIG. 1B, which is at a level above the fluid
level 124 in outlet channel 106. As can be understood, back flow or
diffusion through inlet 114 or outlet 116 can therefore be
inhibited, thus limiting or eliminating cross-interference due to
diffusion of molecules in the fluid from one test site to another
test site.
[0030] A sample fluid may be diluted in dilution channel 110.
Inlets 112 can be conveniently used to dilute the sample fluid
multiple times. The number of inlets 112 may vary, for example,
depending on the sample concentration and the desired amount of
dilution. One of the inlets 112, such as inlet 112A may be adapted
for connection to a sample source (not shown) and the other inlets
such as 112B to 112D may be adapted for connection to a dilution
solution source (not shown). Inlets 112 may also be connected to
one or both of a substrate solution source and a washing solution
source (not shown). Outlet channel 106 may be connected to a waste
dispenser (not shown).
[0031] Substrate 102 can be made of a suitable material such as
plastic, ceramic, graphite, glass, rubber, fabric, printed circuit
board, silicon, suitable polymer, or the like. A combination of two
or more of these materials may also be used. Substrate 102 can be
prepared in a suitable manner known to persons skilled in the
art.
[0032] Channels 104, 106, 108, and 110 can each has a width or
depth on the order of 0.02 .mu.m to 2000 .mu.m, depending on the
application. They can have different widths and depths. For
example, inlet channel 104 can have a width of about 200 .mu.m and
a depth of about 50 .mu.m; outlet channel 106 can have a width of
about 300 .mu.m and a depth of about 200 .mu.m; and each test
channel 108 can have a width of about 100 .mu.m and a well depth of
about 150 .mu.m, wherein all depths are relative to the top surface
of substrate 102. As can be appreciated, in this case, the liquid
flow rate is faster in outlet channel 106 than in inlet channel
104, which can be advantageous because it reduces the chance for
fluid to overflow from test channels 108 back into inlet channel
104. Channels 110 is shaped and sized for supplying a fluid to
inlet channel 104 at a desired rate and for effectively diluting
the fluid by a desired ratio. The size of channel 110 can be
readily determined by a person skilled in the art for a given
application.
[0033] Channels 104, 106, 108 and 110 may be formed using
conventional microchip fabrication techniques including etching,
embossing, molding, or the like. For example, a silicon mold can be
fabricated using a soft-lithography method to make a master device
and then a rubbery polymer (such as polydimethylsiloxane (PDMS))
substrate can be formed from the mold.
[0034] Channel surfaces may be coated with a polymeric, metallic,
or ceramic material, or other materials if desired. In particular,
the test site of each test channel 108 may have a surface, such as
bottom surface 118, suitable for immobilizing molecules thereon.
For example, for immobilizing antigens directly onto a bottom
surface 118, the surface can be made of a plastic such as
polyvinyl, polystyrene, or cellulose. Probe molecules (not shown)
may be immobilized at the test site in each test channel 108. Probe
molecules may be immobilized using a conventional immobilization
technique, including physical adsorption, cross-linking,
entrapment, covalent bonding, grafting, or a combination of the
foregoing techniques. Probe molecules can be immobilized with a
robotic nano- or pico-dispenser, or by introducing a fluid
containing the probe molecules to test channels 108. The test
channel surface may be coated with a surface that is suitable for
selectively immobilizing probe molecules. For example, the surface
may be coated with aminopropyltriethoxysilane (APTES). The coated
surface may then be treated with a suitable cross-linker for
capturing the probe molecules. For example, homo-bifunctional
cross-linkers such as glutaraldehyde, or heterobifunctional
cross-linkers such as
1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC) may be applied
to the APTES surface for selectively binding probe proteins.
Immobilization of probe molecules can be optionally performed
during use before detection. After probe immobilization, a thorough
wash of the channels including the test channels can be conducted.
The channel surfaces outside the test sites can be coated with
blocking agents such as gelatin to eliminate non-specific binding
of molecules to these surfaces. The washing and blocking can be
conducted by automatic operation by connecting biochip 100 to a
pumping system.
[0035] A cover (not shown), such as a transparent cover plate, may
be provided on the top of substrate 102 to seal the channels and
the test channels for flow-through operation. The cover can be made
of a glass, plastic, or acrylic sheet. A film with one adhesive
side may be used as the cover. The cover can prevent contamination
during use. A transparent cover may allow detection of optical
signals originated from the test channels 108.
[0036] Other components may be incorporated in or connected to
biochip 100. For example, fluid sources (not shown) such as sample
sources, substrate sources, washing fluid sources may be provided
or linked to dilution channel 110, such as through one or more of
inlets 112. In an alternative embodiment, some fluid sources may be
connected directly to inlet channel 104, bypassing dilution channel
110. Fluid pumps (not shown) and valves (not shown) for controlling
fluid flow in the channels and for supplying fluid to, or
withdrawing fluid from, the channels may be provided. Electrical
interconnects (not shown) and circuitry (not shown) may be provided
for controlling the operation of biochip 100 and/or for measuring
or detecting a signal such as an optical or electrical signal from
the test sites in test channels 108.
[0037] In operation, a fluid, such as a sample fluid containing
target molecules or a washing buffer solution, is introduced into
test channels 108 through inlet channel 106 and inlets 116.
[0038] The sample fluid may be fed into dilution channel 110
through one of inlets 112, such as inlet 112A. Dilution fluids may
be fed into dilution channel 110 through other inlets 112, such as
inlets 112B to 112D, to dilute the sample fluid. The sample fluid
can be diluted multiple times by introducing a dilution fluid at
each one of a number of inlets 112. The dilution ratio at each
inlet 112 can be individually controlled by adjusting the flow
rates at inlets 112. The diluted sample fluid is received in inlet
channel 104 and then in test channels 108. However, when dilution
is not required or desired, a sample solution may be introduced
into test channels 108 through inlet channel 104 without
dilution.
[0039] Overflow of the fluid from the test channels 108 is received
in outlet channel 106. The fluid levels in the inlet, outlet and
test channels are maintained such that the fluid in test channels
108 is below inlet 114 and the fluid level in outlet channel 106 is
below outlets 116. Therefore, back flow through inlet 114 and
outlet 116 are inhibited. As can be appreciated, such fluid levels
can be maintained conveniently when the fluid flows at a greater
rate in outlet channel 106 than in inlet channel 104.
[0040] Different fluids may be introduced into each test channel
108 depending on the detection technique used. The fluids
introduced may include probe solutions, sample solutions, washing
solutions, substrate solutions, solutions containing labelling or
signal generating molecules, and the like, as will be understood by
persons skilled in the art. A continuous fluid flow may be
maintained. Alternatively, fluid flow may be stopped for a certain
period of time to allow certain interactions to occur, such as for
incubation.
[0041] A molecule or molecular interaction is then detected at one,
some or each of the test sites. The molecule detected can be a
target molecule, or a signal generating molecule for indirect
detection of the target molecule. The molecule or molecular
interaction can be detected using various techniques, including
conventional detection techniques for detecting molecules and
molecular interactions. For example, the molecule or molecular
interaction may be detected by detecting an optical, electrical,
magnetic, radiation or electromagnetic signal indicative of the
presence of the target molecule or occurrence of the molecular
interaction. Example detection techniques include enzyme
immunoassay (EIA) techniques. An EIA technique can be performed
with an electrochemical detection technique (electrochemical EIA or
EIA-EC) or with an optical detection technique. Possible EIA
techniques include enzyme-linked immunosorbent assay (ELISA),
direct EIA, competitive EIA, competitive inhibition EIA, sandwich
EIA, and the like. These EIA techniques can be carried out in
manners known to a person skilled in the art. In such techniques,
the signal generating molecules can be product molecules produced
from enzyme-catalysed reactions, which may be mobile in a fluid.
The enzyme-catalyzed interaction or its product can be detected.
For example, the interaction may generate heat, light, radiation,
or sound. The reaction product may be electrochemically active or
fluorescent. Detection of the enzyme-catalyzed interaction or its
product can thus indicate the presence of the target molecules. For
example, when the products are fluorescent, detection of
fluorescent light from a test channel can indicate the presence of
the target molecules in the sample solution. It can then be
determined that the target molecules are present in the sample
solution. The intensity of the fluorescent light can also indicate
the concentration of the target molecules in the sample solution.
Since back flow or diffusion is inhibited, cross-interference due
to diffusion of these product molecules can be avoided or
reduced.
[0042] The target molecule may be captured by probe molecules
immobilized at the test site. Probe molecules may be
pre-immobilized or immobilized during use. Different probe
molecules may be immobilized at different test sites for detecting
different target molecules.
[0043] For instance, probe proteins can be pre-immobilized on the
bottom 118 of a test channel 108, thus defining the test site.
[0044] If the target protein allows for enzyme labelling, a direct
EIA or ELISA detection may be conducted. In a direct EIA technique,
the probe proteins can selectively capture target molecules, which
are labelled with enzymes, in a sample fluid introduced into the
test channels. A substrate fluid can be then introduced into the
test channel. The substrates in the substrate fluid can produce
detectable products in reactions catalysed by the enzymes attached
to the target molecules at a test site. The higher the
concentration of target molecules in the sample fluid, the more
target molecules will be captured and thus producing a stronger
signal.
[0045] In a competitive ELISA technique, the probe molecules can
selectively capture a specific protein in a complex, which can
contain either a target protein or an enzyme-conjugated competing
protein. A sample fluid containing unknown concentrations of target
molecules can be first introduced into a test channel 108. The
target molecules are captured at some of the binding sites. A fluid
containing known concentrations of competing proteins is then
introduced into test channel 108. The competing proteins are
captured at the remaining binding sites. By detecting how many
competing proteins are captured, the presence or concentration of
the target proteins can be indirectly detected.
[0046] In a competitive inhibition EIA technique, the probe
molecules immobilized on the bottom surface of a test channel 108
can be the same type of molecules as the target molecules, such as
the same antigens. During detection, both a sample fluid and a
fluid containing enzyme-labelled molecules such as antibodies of a
known concentration can be introduced into the test channel. An
enzyme-labelled molecule can bind to either a target molecule or a
probe molecule. There is thus competition between target molecules
and probe molecules for binding with the enzyme-labelled molecules.
Enzyme-labelled molecules attached to the target molecules cannot
interact with the probe molecules. The presence of target molecules
at the test site can inhibit a signal that would have resulted from
interactions between enzyme-labelled molecules and probe molecules.
Thus, the intensity of a detected signal can be inversely
proportional to the concentration of the target molecules in the
test channel. This technique can be particularly useful for
detection of small proteins without a second epitope for enzyme
labels.
[0047] In a sandwich-ELISA technique, the target protein may be
captured by the probe protein. The test channels may be washed
after introducing the sample fluid. Next, a fluid containing some
other proteins labelled with enzymes may be introduced into the
test channel to allow the other proteins to bind to the target
proteins. After incubation, the test channels may be washed before
conducting the ELISA detection. As can be appreciated, a capture
target protein is sandwiched between two proteins, the probe
protein and the enzyme-labelled protein.
[0048] The captured molecules or proteins including target
molecules, such as antibodies or antigens, can be detected, for
example, after introducing a fluid containing substrate molecules
that can be converted by enzymatic reactions to coloured products
for optical detection or electrochemically active products for the
electrochemical detection.
[0049] As mentioned above, after a fluid is introduced into test
channels 108, or after certain reactions are allowed to occur, the
channels may be washed with a washing fluid, which may be fed into
inlet channel 104 through dilution channel 110.
[0050] As can be appreciated by persons skilled in the art, samples
containing same or different types of target molecules may be
transported into different test channels 108 for simultaneous
detection of multi-analytes when the different test channels 108
have different probe molecules immobilized therein.
[0051] In other techniques, the target molecules may be detected
without an enzyme attached thereto. For example, the target
molecules may be labeled with radioactive tracers and detected by
sensing a radiation from each test channel 108. In yet other
techniques, the target molecules may be detected without a label.
For example, certain interaction of a specific molecule with
another molecule may produce detectable heat or light.
[0052] In any event, advantageously, back diffusion can be
inhibited or limited, and multiple sample tests can be
simultaneously performed on biochip 100.
[0053] Further, biochip 100 can be conveniently used for analysis
of both low and high concentration samples. For high concentration
sample, the sample fluid can be conveniently diluted on biochip
100. Saturation in the test channels can be avoided even when the
sample initially has very high concentrations of target molecules.
Dilution can be carried out in multiple steps thus reducing the
concentration at each step. As can be understood, when the dilution
ratio is very high in a single dilution step, large errors can
result. Thus, diluting at a low dilution ratio at each step can
reduce the errors than may result from dilution. Dilution on
biochip 100 can be carried out automatically.
[0054] FIGS. 2A and 2B illustrate a microfluidic biochip 200,
exemplary of another embodiment of the present invention, which has
an X-Y addressable array of test sites. Microchip or biochip 200
has a substrate 202 and inlet, outlet and test channels 204, 206,
and 208 formed therein. Test channels 208 are arranged in rows and
columns. Multiple columns of test channels 208 are formed in
substrate 202. The number of test channels 208 may vary and can be
selected depending on the particular application and manufacturing
considerations. Each column of test channels 208 are separated into
multiple rows by sidewalls 210 and share a common inlet channel 204
and a common outlet channel 206. Rows of test channels 208 in
different columns are aligned for convenient addressing. One test
site is formed in each test channel 208, which comprises a pair of
electrodes 212, one of which is a working electrode and the other a
counter or counter/reference electrode. As can be appreciated,
counter electrodes may be replaced by reference electrodes.
Alternatively, a reference electrode may be added in each test
channel 208. As depicted, electrodes 212 are formed on the opposite
sidewalls 210 within each test channel 208.
[0055] As can be better seen in FIG. 2B, each channel 204, 206 and
208 has a substantially flat bottom 214, 216 or 218. Bottom 214 is
elevated from bottom 218, which is in turn elevated from bottom
216. Therefore, as with microchip 100, back diffusion from one test
site to anther test site can be inhibited in microchip 200. The
respective amount of elevation can be readily determined by persons
skilled in the art depending on the application. For example, each
elevation may vary between 25 and 100 .mu.m. However, in an
alternative embodiment, the channels may also be structured similar
to that of microchip 100. In particular, a well may be formed in
each test channels.
[0056] The detection of molecules or molecular interactions at each
test site can be conducted using electrodes 212. As indicated in
FIG. 2A by dot-dashed lines, the working electrodes 212 in each row
and column can be respectively interconnected. The counter
electrodes 212 in each row and column can be respectively
interconnected. Each row of electrodes can have an X address, such
as X1 to X4, and each column of electrodes can have a Y address,
such as Y1 to Y4. Thus, the electrodes in each test channel can be
addressed with an X-Y addressing technique. Conventional X-Y
addressing techniques such as multiplexing techniques can be used.
Such a technique can be readily implemented by a person skilled in
the art. In microchip 200, electrical interconnections or I/O lines
can be limited with simple multiplexing, as can be understood by a
person of skill in the art.
[0057] The substrate and channels of microchip 200 may be formed as
described above for microchip 100.
[0058] Electrodes 212 may be formed using any suitable technique
including conventional techniques for forming electrodes.
Electrodes 212 may have any suitable shape or size. Different
electrodes may have different shapes and sizes or made of different
materials. Working electrodes in different test channels may be
spaced apart at distances on the order of 0.01 .mu.m to 1000 .mu.m,
depending on the application and manufacturing limitations.
Electrodes 212 may be made of a solid or porous material such as
gold, silver, platinum, copper, titanium, chromium, aluminum, metal
oxide, metal carbide, carbon, graphite, fullerene, conductive
plastic, conductive polymer, metal impregnated polymers, or the
like. A combination of two or more of these materials can be
used.
[0059] Probe molecules (not shown) may be immobilized on working
electrodes 212 or on bottoms 218 of test channels 208 proximate
electrodes 212.
[0060] A cover (not shown), such as a transparent cover plate, may
be optionally provided on the top of the substrate to cover the
channels for sealing the flow-through channels.
[0061] Other components may be incorporated in or connected to
biochip 200. For example, fluid sources (not shown) such as sample
sources, substrate sources, washing solution sources may be
provided or linked to input channel 204. Fluid pumps (not shown)
and valves (not shown) for controlling fluid flow in the channels
and for supplying fluids to the channels may be provided.
Electrical interconnects (not shown) and circuitry (not shown) may
be provided for biasing electrodes 212 and for measuring electrical
signals such as voltages and currents at electrodes 212.
[0062] Biochip 200 may be operated in a similar manner as for
biochip 100. Conveniently, an electrical signal can be sensed using
electrodes 212 for detecting molecules or molecular interactions at
each test site.
[0063] For example, the reaction products of the molecular
interactions may comprise electrochemically active molecules. Each
pair of electrodes 212 in a test channel 208 is biased to monitor
the electrochemical reactivity at the test site in the test
channel. As can be appreciated, when electrochemically active
products are present in the liquid in the test channel, an
electrical current can flow between the pair of electrodes 212.
[0064] As can be understood, in an EIA technique, a highly
concentrated substrate solution can be used to produce a high
concentration of electrochemically active products even when the
target molecules have a very low concentration in the sample
solution. Thus, such a technique can be highly sensitive.
Alternatively, the product concentration can be significantly
increased for high sensitivity detection by increasing the
incubation time for the enzymatic reaction.
[0065] The amount of electrochemically active products produced at
a test site can be detected by monitoring an electrical signal at
electrodes 212, such as a current through an electrode 212. The
concentration of the target molecules can be determined, for
example, by comparing the detected current with a calibration curve
of current vs. concentration, which can be obtained by measuring
samples with known concentration of target molecules. In an
amperimetric approach, the current due to generation of the
electrochemically active products produced from the enzymatic
reaction can be proportional to the concentration of the product,
and can be approximately proportional to the target concentration
in the sample solution.
[0066] As can be understood, as the test sites are separated by the
sidewalls 210 and back flow from outlet channels 206 into test
channels 208 and from test channels 208 into inlet channels 204 can
be inhibited, diffusion of molecules from one test site to another
can be limited or inhibited. In addition, when a liquid flow is
maintained in the channels, upstream diffusion of molecules within
test channels 208 is further limited. If the liquid can flow faster
in outlet channel 206 than in inlet channel 208, backflow from
outlet channel 206 into a test channel 208 can be further limited.
As such, the chances of products produced at one test site moving
to another test site can be reduced.
[0067] To guide fluid towards electrodes 212, a section of each
test channel 208 proximate the test site, i.e. electrodes 212, may
be narrowed, as illustrated in FIGS. 3A and 3B.
[0068] FIGS. 3A and 3B illustrate a biochip 300 which is a
variation of biochip 200. As in biochip 200, biochip 300 includes a
substrate 302, and an inlet channel 304, an outlet channel 306, and
a plurality of test channels 308 formed therein. Inlet channel 304
has a bottom surface 314. Outlet channel 306 has a bottom surface
316. Test channels 308 are defined by sidewalls 310 and bottom
surface 318.
[0069] Biochip 300 has a panel 320 extending between sidewalls 310
and elevated from bottom surface 318, thus defining an opening 322
to allow the liquid to flow through. The narrowed section at
opening 322 guides fluid towards the test site for effective
contact with electrodes 212 or a surface having probe molecules
immobilized thereon. Panel 320 can also further prevent backflow.
Panel 320 may be shifted vertically to vary opening 322 for
controlling the flow rate.
[0070] Electrodes 212 may be disposed in a test channel differently
than as shown in FIGS. 3A and 3B. For example, as illustrated in
FIG. 4, electrodes, such as electrodes 412, can be formed on
bottoms 318 of test channels 308. A pair of working and
counter/reference electrodes 412 may be disposed at bottom 318
adjacent opposite sidewalls, as depicted.
[0071] As discussed above, probe molecules can be immobilized on a
surface in a test channel. Probe molecules may include antibodies,
antigens, peptides, aptomers, DNAs, and the like, which can
specifically capture and bind to the target molecules. Probe
molecules can capture target molecules contained in a sample
solution introduced into the test channels. The sample solution may
be flown through each test channel to allow the target molecules
and/or enzyme-labelled molecules (reporter molecules) to be
captured by the probe molecules. The reporter molecules in a
competition detection scheme are labelled with enzyme molecules. A
reporter molecule can be an antibody, antigen or any molecule with
affinity to the probe molecule. The target molecules may be
directly labelled before they react with the probe molecules. In
each of the above steps, a liquid may be flown through the channels
to attach a next molecule to the already immobilized molecule. To
ensure accuracy and efficiency, the channels may be washed with a
washing solution before feeding the next liquid. In particular, the
channels may be washed before providing substrate solutions to the
test channels to ensure that mobile enzymes are removed from the
test channels.
[0072] A specific example of a sandwich immunoassay technique is
illustrated in FIG. 5A. A probe antibody is immobilized on the
surface of a working electrode or surface close to the working
electrode. The target molecule is an antigen of interest and is
bond to the probe antibody. An enzyme (such as ALP)-labelled second
antibody is transported to bind to the target antigen after
washing. Then the substrate, such as a PAPP including
dephosphorylase-PAPP, is delivered into the detection spots for an
enzymatic reaction, for example by flowing a substrate solution
through the test channels. The substrate molecule reacts under
enzyme catalysis at the enzyme active site to produce an
electrochemically active product, PAP. Advantageously, PAP can be
detected electrochemically when the electrodes are biased to a low
potential such as 300 mv with reference to an Ag/Ag reference
electrode.
[0073] Similarly, in the example shown in FIG. 5B, a probe DNA is
first immobilized on the electrode surface. The target DNA is then
bond to the probe DNA. The target DNA is labelled with an ALP
enzyme. The enzyme can catalyse conversion of PAPP substrates to
PAP products as in FIG. 5A for electrochemical detection.
[0074] FIG. 6A shows a partial image of a silicon mold for forming
a biochip exemplary of embodiments of the present invention. The
mold was prepared with a soft-lithography method. The test channels
(seen as parallel narrow channels) have widths of about 100
.mu.m.
[0075] FIG. 6B shows a partial image of a biochip formed from the
mold shown in FIG. 6A. The biochip was formed by pouring a rubbery
polymer (PDMS) over the mold, curing the polymer, and then
separating the cured polymer from the mold. The biochip is suitable
for ELISA analysis or detection. Probe antigens were immobilized in
the test channels. A transparent polyacrylic sheet was attached to
the biochip as the cover for sealing the device.
[0076] To test biochips such as the one shown in FIG. 6B, target
proteins were immobilized as follows on the biochips. A thin
aminopropyltriethoxysilane (APTES) film was coated or grafted on
the PDMS surface of the test channels. The APTES surface was
treated with homo-bifunctional cross-linkers (glutaraldehyde) for
capturing immunoglobulin proteins on the APTES film. Alternatively,
succinic acid anhydride generated carboxyl groups were applied onto
the APTES surface, and heterobifunctional cross-linkers
(1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC)) were then
applied to the surface to covalently bind proteins onto the
PDMS-APTES surface through the carboxyl groups.
[0077] A PDMS-APTES surface can be prepared by first cleaning the
PDMS surface and then immersing the surface in an ethanol solution
containing about 10% v/v APTES for about 10 minutes at room
temperature. The APTES treated PDMS is rinsed with 96% ethanol,
followed by air-drying, and then heated at 80.degree. C. in a
vacuum oven for about 2 hours.
[0078] Covalent immobilization can be realized via EDC as follows.
Succinic acid, anhydride (SAA) can be first used to modify the
APTES surface by immersing the PDMS-APTES surface in a 50 g/ml SAA
solution for about 2 hours at room temperature. The pH value of the
solution is adjusted by using a 3M NaOH solution to keep the
solution stable at pH 6.0. Then surface is rinsed by
phosphate-citrate buffer and dried under nitrogen flow. EDC and
protein mixture are dropped onto the modified solid surface for
cross-linking. The concentration of the EDC is 20 .mu.g/ml. The
reaction buffer has 0.01M of phosphate-citrate, with a pH of 4.6.
The cross-linking process lasts for about 1 hour at room
temperature. The cross-linking process is terminated by immersing
the surface in a Tris-HCl buffer. The protein coated surface is
then immersed in a 1% bovine serum albumin (BSA) for blocking
unoccupied surface for about 2 hours at about 37.degree. C. or for
about 8 hours at room temperature.
[0079] Covalent immobilization can also be realized via
glutaraldehyde (GA) as follows. The APTES treated PDMS surface is
modified by GA to covalently immobilize protein on the PDMS
surface. The amino groups on the surface are activated by 2.5% GA
for 1 hour at room temperature. The surface is rinsed with a pH 8.0
Tris-HCl buffer, and dried with nitrogen gas. Protein probes in
Tris-HCl are added onto the glutaraldehyde-activated surface. The
cross-linking reaction can take place at room temperature for about
2 hour. After washing, the protein-coated surface is
surface-blocked with the block reagent described above.
[0080] Example biochips fabricated as above have shown good
performance. For example, rabbit IgG were immobilized in the test
channels as described in the preceding paragraph. Sample solutions
containing varying concentrations of enzymes labeled with
anti-rabbit IgG were flown through the channels and test channels
to immobilize the enzymes in the test channels by antibody-antigen
binding. After feeding each sample solution, the channels and test
channels was washed with a washing solution. A substrate solution
was then flown through the test channels. Fluorescent light emitted
from the test channels was detected. The intensities of the
fluorescent light were substantially linearly dependent on the
concentration of the sample solutions. The linear dependence
indicates that cross-interference between test channels was very
low. It has been found that as low as about 5.6 pg/ml of enzyme
concentration can be detected.
[0081] Exemplary embodiments disclosed herein are useful for
detecting low level of antigens, antibodies, peptides, DNA,
proteins and other biomolecules in fluid or tissue samples. Low
levels of DNA, RNA and other biomolecules such as glucose, which
can be enzymatically converted to electrochemically active species,
can be detected using embodiments of the present invention.
Embodiments of the present invention can be incorporated into
existing optical detection systems based on enzyme-catalyzed
bio-reactions, in which PCR amplification can be eliminated. The
exemplary biochips disclosed herein may provide a basic platform
for a variety of applications, for example in diagnostics, drug
discovery, target validation and pathogen detection.
[0082] Embodiments of the invention can be used in competition and
displacement immunoassays where target antigens are labeled with
enzymes for competition binding or displacement for detection.
Radioactive tracers can be used to label the target molecules or
binding molecules as reporters. The sandwich immunoassay is
suitable for use with high molecular weight antigens, which possess
at least two antigenic determinants or epitopes, but the
competition and displacement immunoassay can be used to detect
different antigens.
[0083] As can be appreciated, the operation of the biochips
described herein can be automated. The biochips can be connected to
utility modules. The operation of a biochip can be controlled by a
computer or a microprocessor. The liquid flow rate and flow time
can be conveniently adjusted.
[0084] As the substrate solution can continuously flow through the
test sites generating more and more product molecules, even a low
concentration of target molecules in the analyte solution can be
detected. The substrate solution can also have a high concentration
of substrate molecules. The liquid flow rate and flow time can be
conveniently adjusted to control the enzyme reaction, for example,
for achieving different detection sensitivities.
[0085] As can be appreciated, target molecules can be immobilized
or labelled in other suitable manners. For example, the surface of
an electrode may have a coating that can selectively capture the
target molecules. In a further example, when the target molecule is
itself an enzyme, it may not be necessary to attach another enzyme
to it.
[0086] As can be understood, the enzyme reactions or target
molecules present at different test sites can be identified using
electrode addressing techniques including conventional addressing
techniques. For example, the test sites may form an array of rows
and columns and X-Y addressing techniques may be used.
[0087] Advantageously, high-density array biochips disclosed herein
can be made compact without creating substantial cross-interference
for a flow-through system. It can also be fabricated inexpensively
and easily, for example, using inexpensive and matured printed
circuit board technology to fabricate a plastic based microarray
chip with microchannels, or embossing fabrication process.
[0088] As can be appreciated, working and counter or reference
electrodes 212 may be interchangeable. In alternative embodiments,
separate counter and reference electrodes, thus more than two
electrodes, can be provided in each test channel 208. The reference
electrodes can be interconnected with each other. The
implementation and operation of electrodes for detecting
electrochemical reactions including the use of reference electrodes
can be readily understood by persons skilled in the art.
[0089] Embodiments of the invention may also be useful in protein
expression profiling. Measurement of the variation in the
expression of known proteins within tissues or cells over time, or
in response to challenge by drugs, toxins, injury or disease, may
require high throughput operation and can be conveniently performed
using embodiments of the present invention.
[0090] Embodiments of the invention can be used in high density
array-sensors with high sensitivity and specificity. Accuracy and
reliability of the embodiments disclosed herein can be high because
cross-interference due to diffusion can be inhibited or limited. It
is also convenient to perform multi-fold dilution of sample,
automatic post-immobilization probe treatment, and washing.
[0091] Advantageously, array dispensers are not necessary for
operating biochips disclosed herein. Total assay time can be
reduced compared to conventional biochips. The requirement of the
samples amount is significantly decreased.
[0092] Other features, benefits and advantages of the present
invention not expressly mentioned above can be understood from this
description and the drawings by those skilled in the art.
[0093] The above described embodiments are intended to be
illustrative only and in no way limiting. The described embodiments
are susceptible to many modifications of form, arrangement of
parts, details and order of operation. The invention, rather, is
intended to encompass all such modification within its scope, as
defined by the claims.
* * * * *